-Draft OECD GUIDELINE FOR TESTING OF CHEMICALS

OECD/OCDE Draft: 21 December 2020 | 1

-Draft OECD GUIDELINE FOR TESTING OF CHEMICALS 1

In Vitro Phototoxicity: Reconstructed Human Epidermis Phototoxicity 2

Test (RhE PT) Method 3

4

INTRODUCTION 5 6

1. Phototoxicity (photoirritation) is defined as an acute toxic response elicited by topically or 7

systemically administered photoreactive chemicals after the exposure of the body to 8

environmental light. Within the context of skin exposures to phototoxic chemicals, 9

phototoxic responses are elicited after the first exposure of skin to photoactive chemicals 10

and subsequent exposure to light. 11

12

2. This Test Guideline addresses the human health endpoint of phototoxicity, specifically as it 13

relates to topical skin exposures to phototoxic chemicals. The in vitro reconstructed human 14

epidermis phototoxicity test (RhE PT) is used to identify the phototoxic potential of a test 15

chemical after topical application in reconstructed human epidermis (RhE) tissues in the 16

presence and absence of simulated sunlight. Phototoxicity potential is evaluated by the 17

relative reduction in viability of cells exposed to the test chemical in the presence as 18

compared to the absence of simulated sunlight (see paragraphs 36-37 for the 19

characterization of simulated sunlight). Chemicals identified as positive in this test may be 20

phototoxic in vivo following topical application to the skin, eyes, and other external light-21

exposed epithelia. 22

23

3. This Test Guideline is based on the in vitro test system of the reconstructed human 24

epidermis (RhE), which closely mimics the biochemical and physiological properties of the 25

outermost layers of the human skin, i.e., the epidermis. The RhE test system uses human-26

derived non-transformed keratinocytes as a cell source to reconstruct an epidermal model 27

with representative histology and cytoarchitecture. 28

29

4. An assessment of the general performance was based on an ad hoc evaluation of individual 30

literature citations (1) including an initial test method pre-validation reported in 1999 (2) 31

with a sensitivity of 86.7% and specificity of 93.3%. Mutual Acceptance of Data will only 32

be guaranteed for test methods that are validated according to the Performance Standards 33

and have been reviewed and adopted by OECD. The test methods included in this TG can 34

be used indiscriminately to address countries’ requirements for test results from in vitro test 35

methods for phototoxicity while benefiting from the Mutual Acceptance of Data. 36

37

5. Definitions used in this Test Guideline are provided in Annex 1. 38

39

INITIAL CONSIDERATIONS AND LIMITATIONS 40 41

6. Many types of chemicals have been reported to induce phototoxic effects (3)(4)(5)(6). Their 42

common feature is their ability to absorb light energy within the sunlight emission 43

spectrum. Photoreactions require sufficient absorption of light quanta. Thus, before testing 44

is considered, a UV/visible absorption spectrum of the test chemical should be determined 45

according to OECD Test Guideline 101. It has been reported that if the molar 46

extinction/absorption coefficient (MEC) is less than 1000 L mol-1 cm-1, the chemical is 47

unlikely to be photoreactive (7)(8). Such chemicals may not need additional testing with the 48

in vitro RhE PT or any other biological test for adverse photochemical effects (1)(9). In 49


general, this principle applies to all test chemicals, however, more specific guidelines may 50

apply depending on the intended use of the chemical or potential exposure conditions. The 51

RhE PT test can be used as a stand-alone method, and also in a tiered testing strategy for 52

topically applied substances following specific guidelines (such as ICH S10 for 53

pharmaceuticals). See also Annex 2 for guidance on testing the phototoxicity potential of a 54

formulation or complex mixture at “end-use” concentrations. 55

56

7. The reliability and relevance of the in vitro RhE PT was evaluated in multiple studies (1). 57

The procedures and prediction model presented in this test guideline are designed to 58

distinguish between phototoxic and non-phototoxic compounds. However, specific 59

procedures and prediction models exist in the literature to address phototoxic potency for 60

topically applied compounds and mixtures. This test is not designed to predict other adverse 61

effects that may arise from the combined action of a chemical and light (e.g., it does not 62

address photo-genotoxicity, photoallergy, or photocarcinogenicity). Furthermore, the test 63

has not been designed to address indirect mechanisms of phototoxicity, effects of 64

metabolites of the test chemical, or evaluate the phototoxic potential of individual 65

chemicals in mixtures. 66

67

8. The in vitro RhE PT does not need to be performed with a metabolic activation system, 68

although the RhE tissues have limited metabolic activity (10). There is no evidence at this 69

time that any phototoxic compound would be missed in the absence of metabolic activation 70

(11). 71

72

9. Test chemicals absorbing light in the same range as MTT formazan (colored chemicals), or 73

test chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere 74

with the cell viability measurements if those chemicals persist in or on the test system at the 75

time of the viability assessment, and may need to use adapted controls to correct for the 76

interference (see paragraphs 58-65) in section “Corrections for MTT-reducing Materials 77

and Colorants”. 78

79

10. Although most of the studies performed with RhE PT utilized the UVA/visible light part of 80

the solar spectrum, some studies confirm that the RhE tissues can also tolerate UVB 81

exposure under controlled conditions. This is an advantage compared to most of the cell-82

line based assays (OECD TG 432) that do not tolerate the UVB part of the spectrum well 83

(12)(13). 84

85

11. A single testing run should be sufficient for a test chemical when the classification is 86

unequivocal. However, in cases of borderline results, such as non-concordant results from 87

replicate tissues, a second run should be considered, as well as a third one in case of 88

discordant results between the first two runs. In the repeated runs, the concentrations of the 89

test chemical may be adjusted to better capture the range of responses around the borderline 90

or equivocal concentration(s). 91

92

12. The phototoxicity potential of a test chemical is determined by testing multiple 93

concentrations in RhE tissues in the presence and absence of simulated sunlight. The testing 94

of three to five concentrations is generally sufficient to ensure obtaining acceptable test 95

results from at least one concentration of the test chemical to make a valid prediction. 96

Specific criteria for acceptable test results are presented with the Prediction Model. 97

98

PRINCIPLE OF THE TEST 99 100

13. Phototoxicity potential in the RhE-PT is evaluated by the relative reduction in viability in 101

RhE tissues exposed to the test chemical in the presence as compared to the absence of a 102

non-cytotoxic dose of simulated sunlight. 103


104

14. The test chemical is applied topically to a three-dimensional RhE tissue, composed of non-105

transformed human-derived epidermal keratinocytes that have been cultured to form a 106

multilayered, highly differentiated model of the human epidermis. It consists of organized 107

basal, spinous and granular layers, and a multilayered stratum corneum containing 108

intercellular lamellar lipid layers representing main lipid classes analogous to those found in 109

vivo. Accordingly, RhE tissues are ideally suited for directly modeling exposures of 110

chemicals on native skin in vivo and have been validated to predict the skin irritation and 111

corrosion hazards of chemicals and mixtures without the need for test chemical dilution 112

(24)(25). 113

114

15. In brief, several concentrations of test chemical prepared in a solvent are applied topically 115

to RhE tissues and incubated at standard culture conditions (37 ± 1 °C, 5 ± 1% CO2, 116

90 ± 10% RH) for 18 to 24 hours to allow penetration into the living tissue. In general, three 117

to five concentrations are tested to ensure obtaining results from at least one concentration 118

that meets the criteria for a valid test. A positive control and appropriate solvent controls are 119

also applied topically to RhE tissues and tested in parallel. Half of the tissues in each 120

treatment group are irradiated with 6 J/cm2 of simulated sunlight (+Irr) while the remaining 121

half are held at room temperature in the dark (−Irr). After a post-exposure incubation period 122

of 18 to 24 hours, relative viability is determined in both the irradiated (+Irr) and non-123

irradiated (−Irr) treatment groups by measuring the enzymatic conversion of the vital dye 124

MTT (3-[4,5 dimethylthiazol 2 yl] 2,5-diphenyltetrazolium bromide, thiazolyl blue (CAS 125

number 298-93-1) into a blue formazan salt that is measured photometrically after 126

extraction from the tissues. 127

128

16. Phototoxic potential is determined by comparing the relative reduction in viability in each 129

irradiated treatment group to that of the equivalent non-irradiated treatment group. 130

131

17. The experimental design is based on the pre-validation study performed by ZEBET (2)(14) 132

and follow up-studies conducted with this protocol. The follow-up studies suggested some 133

minor modifications that led to better reproducibility and sensitivity of the test. The updated 134

protocol was published in 2018 (15). 135

136

18. This test method can be used as a stand-alone test method to address phototoxicity, 137

especially in cases of limited test material solubility or endpoint-compatibility issues with 138

the 3T3 Phototoxicity Test (OECD TG 432) and ROS Assay for Photoreactivity (OECD TG 139

495). This test method can be used in a tiered testing strategy in combination with the 140

OECD TG 432 and/or OECD TG 495 (11)(27)(28). Since the test system incorporates the 141

skin barrier function of the RhE tissues, with appropriate justifications, complex 142

formulations may also be tested at “end-user” concentrations or as a neat application to 143

evaluate for phototoxic potential (see Annex 2 for guidance). 144

145

DEMONSTRATION OF PROFICIENCY 146

147 19. Prior to the routine use of the test method, laboratories should demonstrate technical 148

proficiency, using the Proficiency Substances listed in Table 1. In situations where a listed 149

chemical is unavailable or cannot be used for other justified reasons, another chemical for 150

which adequate in vivo and in vitro reference data are available may be used (e.g., from the 151

list of reference chemicals (1)) provided that the same selection criteria as described in 152

Table 1 are applied. Using an alternative proficiency substance should be justified. 153

154

20. As part of the proficiency testing, if users are naïve to utilizing the RhE model within the 155

testing facility, it is recommended that users verify the barrier properties of the tissues after 156

receipt as specified by the RhE model producer. This is particularly important if tissues are 157


shipped over long distance/time periods. However, once a test method has been successfully 158

established and proficiency in its use has been demonstrated, such verification will not be 159

necessary on a routine basis. 160

161

162

163

164

Table 1. Proficiency Substances1 165

166

Substance CAS RN In vivo2 Solvent3

Typical phototoxicity ranges

[% w/v or % v/v]

(references)

PHOTOTOXIC SUBSTANCES

1 Chlorpromazine 50-53-3 PT Water 0.003% – 0.01%

(2)

2 Anthracene 120-12-7 PT EtOH4 0.01% – 0.03%

(12)(22)

3 Bergamot oil

(non-purified)

8007-75-8 PT Oil5 0.0316% – 3.16%6

(2)(27)

NON-PHOTOTOXIC SUBSTANCES

4 Sodium Lauryl

Sulfate

151-21-3 NPT Water Non-phototoxic up to highest conc. tested (1%)

(2)

5 Octyl salicylate 118-60-5 NPT Oil5 Non-phototoxic up to highest conc. tested (10%)

(2)

6 Butyl Methoxy-

dibenzoylmethane

70356-09-1 NPT EtOH4 or

Oil5

Non-phototoxic up to highest conc. tested (10%)

(12)(28) Notes: 1 The Proficiency Substances are a subset of the substances used in the pre-validation and follow up studies and the 167 selection is based on the following criteria; (i), the substances are commercially available; (ii), they are representative of the 168 full range of phototoxic effects (from non-phototoxic to strong photoirritants); (iii), they have a well-defined chemical 169 structure; (iv), they are representative of the chemical functionality used in the validation process; (v) they provided 170 reproducible in vitro results across multiple testing and multiple laboratories; (vi) they were correctly predicted in vitro, and 171 (vii) they are not associated with an extremely toxic profile (e.g., carcinogenic or toxic to the reproductive system) and they are 172 not associated with prohibitive disposal costs, and (viii) results for the selected materials and protocol details are available in 173 the literature. 174 2 PT – Phototoxic; NPT – Non-Phototoxic 175 3 Solvents are suggested, based upon the pre-validation and follow-up study references 176 4 EtOH – Ethanol 177 5 Oil – Sesame seed oil 178 6 Variability in phototoxic response may be influenced by the content of impurities 179 180

181

182

PROCEDURE 183 184

21. The following is a description of the components and procedures of a RhE test method for 185

phototoxicity testing. Standard Operating Procedures (SOPs) for the RhE-based tests 186

complying with this TG are available (14)(15) 187

188

General Test System Characterisation 189

190

22. Non-transformed human keratinocytes should be used to reconstruct the epithelium. 191

Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum 192

granulosum) should be present under a functional stratum corneum. The stratum corneum 193

should be multi-layered containing the essential lipid profile to produce a functional barrier 194

with robustness to resist rapid penetration of cytotoxic benchmark chemicals (e.g., the 195

surfactants sodium dodecyl sulphate (SDS) and Triton®-X-100 are typically used to test 196

barrier function). The containment properties of the RhE model should prevent the passage 197


of material around the stratum corneum to the viable tissue, which would lead to poor 198

modelling of skin exposure. The RhE tissue should be free of contamination by bacteria, 199

viruses, mycoplasma, or fungi. 200

201

Functional Conditions 202 203

Viability 204

23. The assay used for quantifying viability is the MTT-assay (16). The viable cells of the RhE 205

tissue can reduce the vital dye MTT into a blue MTT formazan precipitate which is then 206

extracted from the tissue using isopropanol (or a similar solvent). The optical density (OD) 207

of the extraction solvent alone should be sufficiently small, i.e. OD < 0.1. The extracted 208

MTT formazan may be quantified using either a standard absorbance (OD) measurement or 209

an HPLC/UPLC-spectrophotometry procedure (17). The RhE model developer/supplier 210

should ensure that each batch of the RhE model meets defined quality control criteria for 211

the negative controls. Acceptability ranges (upper and lower limit) for the negative control 212

OD values are established by the RhE model developer/suppliers and presented in Table 2. 213

The RhE model user should ensure that the results of the solvent (i.e. negative) controls 214

meet the specific test method acceptance criteria. An HPLC/UPLC Spectrophotometry user 215

should use the negative control OD ranges provided in Table 2 as the acceptance criterion 216

for the solvent (i.e. negative) control. 217

218

Table 2. Acceptability ranges for solvent (i.e. negative) control OD values in the MTT assay of the 219

test methods included in this TG 220

221

Lower acceptance limit Upper acceptance

limit

EpiDerm™ (EPI-200) 0.8 2.8

t.b.a.

222

Barrier function 223

24. The RhE model developer/supplier should ensure that each batch of the RhE model meets 224

defined quality control criteria for barrier function. The barrier function may be assessed 225

either by determination of the concentration at which a benchmark chemical (e.g., sodium 226

dodecyl sulphate (SDS) or Triton®-X-100) reduces the viability of the tissues by 50% 227

(IC50) after a fixed exposure time, or by determination of the exposure time required to 228

reduce cell viability by 50% (ET50) upon application of the benchmark chemical at a 229

specified, fixed concentration. The acceptability ranges for the test methods included in this 230

TG are given in Table 3. 231 232

Table 3. Barrier Function QC batch release criteria of the RhE models included in this TG 233

234

Lower acceptance limit Upper acceptance

limit

EpiDerm™ (EPI-200) ET50 = 4.00 h ET50 = 8.72 h

t.b.a.

235

236

Morphology 237

25. Histological examination of the RhE model may be provided by the RhE model 238

developer/supplier demonstrating human epidermis-like structure (including multilayered 239

stratum corneum) if this parameter is used in the RhE model developer/supplier’s QC 240

release program. 241

242


Reproducibility 243

26. The RhE model developer/supplier should maintain a database of the QC release test results 244

of the viability and barrier function tests to monitor reproducibility over time. It is 245

recommended that the RhE model user maintain a database of the phototoxicity test method 246

positive and solvent (i.e. negative) control results to monitor reproducibility of test method 247

execution over time. 248

249

Quality control (QC) 250

27. The RhE model should only be used if the developer/supplier demonstrates that each batch 251

of the RhE model meets defined production release criteria, among which those for viability 252

(paragraph 23), barrier function (paragraph 24) and morphology (paragraph 25), if 253

applicable, are the most relevant. The relevant QC data should be provided to the test 254

method users, so that they are able to include this information in the test report. Only 255

phototoxicity test results produced with qualified tissues can be accepted for reliable 256

prediction of phototoxicity. 257

258

Preparation of Test Chemical and Control Substances 259

260 28. Test chemicals must be prepared fresh on the day of testing unless data demonstrate their 261

stability in storage. It is recommended that all chemical handling and the initial treatment of 262

tissues be performed under conditions that would avoid photoactivation or degradation of 263

the test chemical prior to irradiation. The maximum recommended concentration of a test 264

chemical should not exceed 10% since test chemicals may absorb UV and act as a UV-filter 265

(14)(15). 266

267

29. The testing of three to five concentrations of a test chemical in a solvent is generally 268

sufficient to ensure obtaining acceptable test results from at least one concentration of the 269

test chemical to fulfill requirements to evaluate the test results for phototoxic potential (see 270

paragraphs 68-70). Ideally, the concentrations of the test chemical should be selected to 271

ensure a cytotoxicity dose response in the absence of irradiation. Guidance for selection of 272

appropriate concentration ranges is given in the SOPs (14)(15). 273

274

30. Water soluble test chemicals are prepared in ultra-pure water or if appropriate in buffered 275

salt solutions (e.g., Dulbecco’s Phosphate Buffered Saline (DPBS) or Hanks' Balanced Salt 276

Solution (HBSS) without phenol red). The buffer used must be free from protein 277

components and light absorbing components (e.g., pH indicators such as phenol red and 278

vitamins) to avoid interference during irradiation. 279

280

31. Oil soluble test chemicals are prepared in sesame seed oil or other appropriate oil (e.g., 281

mineral oil that has low UV absorption and is demonstrated to be compatible with the RhE 282

tissues). For test chemicals of limited solubility in water and oils, pure ethanol, or a mixture 283

of acetone:olive oil (4:1 v:v) may be used (15). 284

285

32. Other solvents may be considered but should be evaluated prior to use for specific 286

properties including compatibility with the RhE tissues, its ability to react with the test 287

chemical, ability to induce phototoxicity, potential for quenching of the phototoxic effect, 288

radical-scavenging properties and/or chemical stability in the solvent (18). When other 289

solvents are used, it is recommended that a pre-testing with the selected solvent be 290

conducted to ensure solvent stability and compatibility with the test system (see Annex 4 291

for additional guidance). 292

293

33. Vortex mixing, sonication, and/or warming to appropriate temperatures may be used to aid 294

solubilisation, unless the stability of the test chemical is compromised. The procedures used 295

to prepare the test chemical dosing solutions should be documented. 296


297

34. Before any testing on the viable reconstructed human tissues is performed, it is 298

recommended to perform the evaluation of the test substance for interference with the 299

measured endpoint (MTT assay). These procedures are described in detail in the SOPs. If 300

potential interference by the test substance on the MTT assay has been determined, the 301

application of adaptive controls is recommended as described in the section “Corrections 302

for MTT-reducing Materials and Colorants.” 303

304

35. A solvent control (used as a negative control) and positive control (PC) should be tested 305

concurrently in each run. The suggested solvent control is either water (solvent for water 306

soluble materials) or sesame seed oil (solvent for oil soluble materials), and/or other 307

solvents used to solubilize the test material. The suggested PC is a solution of 308

chlorpromazine at a final concentration of 0.01% to 0.02% in water (or other aqueous 309

buffered salt solutions such as DPBS or HBSS without phenol red). Additional 310

concentrations can be tested to evaluate dose responses of the chlorpromazine prior to 311

establishing the test to demonstrate proficiency (1)(14)(15). 312

313

Irradiation Conditions 314 315

36. Light source: The choice of an appropriate light source (e.g., a solar simulator) and filters 316

is a crucial factor in phototoxicity testing. Light of the UVA and visible regions is usually 317

associated with phototoxic reactions in vivo (5)(19), whereas generally UVB is of less 318

relevance but is highly cytotoxic; the cytotoxicity increases 1000-fold as the wavelength 319

goes from 313 to 280 nm (20). Acceptable light sources emit the entire solar spectrum (290 320

nm through 700 nm). Adjustment of the spectrum can be performed using filters to 321

attenuate UVB while allowing transmittance of UVA and visible light (See Annex 3). 322

Furthermore, the wavelengths, irradiance doses employed, and light source equipment (e.g., 323

open or closed system) should not be unduly deleterious to the test system (e.g., from 324

emission of heat/ wavelengths in the infrared region). 325

326

37. The simulation of sunlight with solar simulators is considered the optimal artificial light 327

source. The spectral irradiance of the filtered solar simulator should be close to that of 328

outdoor daylight (21). Both xenon arcs and (doped) mercury-metal halide arcs are used as 329

solar simulators (22). The latter have the advantage of emitting less heat and being cheaper, 330

but the match to sunlight is not as good as that provided by xenon arcs. All solar simulators 331

emit significant quantities of UVB and should be suitably filtered to attenuate UVB 332

wavelengths (Annex 3). Because cell culture plastic materials contain UV stabilisers, the 333

transmitted spectrum should be measured through the same type of plate lid as will be used 334

in the assay. Irrespective of measures taken to attenuate parts of the spectrum by filtering or 335

by unavoidable filter effects of the equipment, the spectrum recorded below these filters 336

should not deviate from standardised outdoor daylight (21). External light standard D65, the 337

internationally recognized emission standard for outdoor daylight, is provided in ISO DIS 338

18909:2006. An example of the spectral irradiance distribution of the filtered solar 339

simulator used in pre-validation and follow-up studies with the EpiDerm™ model is given 340

in (14)(15)(22). See also Annex 3 Figure 1. 341

342

38. Dosimetry: The intensity of light (irradiance) should be regularly checked before each 343

phototoxicity test using a suitable broadband UVA-meter. Irradiance should be measured 344

through the same type of plate lid as will be used in the assay. 345

346

39. An irradiance dose of approximately 6 J/cm2 (as measured in the UVA range) was 347

determined to be non-cytotoxic in the RhE tissues and sufficiently potent to excite 348

chemicals to elicit phototoxic reactions (2)(15)(22). To achieve 6 J/cm2 within a time period 349

of 60 minutes, irradiance was adjusted to 1.7 mW/cm2 of UVA/visible light (see Annex 3, 350


Figure 2). Alternate exposure times and/or irradiance values may be used to achieve 6 J/cm2 351

using the formula: 352

353

354

355

356

357

358

40. The RhE tissue model is tolerant to UVB irradiation and inclusion of UVB irradiation may 359

be appropriate in some cases (e.g., when absorption peaks for the test chemical of interest 360

are exclusively in the UVB wavelength regions). The presence of the UVB portion of the 361

spectra should be monitored and reported in the final report along with any changes in 362

irradiance. 363

364

41. Similarly, if another RhE model or a different light source is used, the irradiation should be 365

calibrated so that a dose regimen can be selected that is not deleterious to the cells but 366

sufficient to excite standard phototoxins. A functional check should be performed by 367

testing the proficiency chemicals described in Table 1 and also presented in Figure 2. 368

369

42. Radiation sensitivity of the cells: A UVA-sensitivity experiment should be performed once 370

the test is newly set up in a laboratory. A brief description of the method and expected 371

outcome is given in the SOPs. The viability of the irradiated tissues exposed to 6 J/cm2 372

should be ≥ 80% relative to the tissues that were not irradiated. 373

374

375

Test procedure 376

377

43. Tissue conditioning: Upon receipt of the RhE tissues, examine all kit components for 378

integrity. Under sterile conditions, transfer tissues to 6-well plates containing 0.9 mL 379

medium/well. Place the plates into the incubator at standard culture conditions (37 ± 1 °C, 380

5 ± 1% CO2, 90 ± 10% RH) for minimum of 60 minutes. Pre-incubation can be extended 381

overnight, however the medium should be exchanged after the first 60 minutes. 382

383

44. After a minimum of 60 minutes pre-incubation, the tissues in the 6-well plates will be 384

removed from the incubator and the medium under the tissues will be exchanged with 385

warmed (37 ºC) fresh assay medium. The tissues may be dosed immediately, or placed back 386

into the incubator until dosing is initiated. For each treatment condition or treatment group, 387

four tissues will be treated such that two tissues are used in the cytotoxicity part of the assay 388

(treated in the absence of irradiation) and two are used in the phototoxicity part of the assay 389

(treated in the presence of irradiation). 390

391

45. Dose Application: The RhE tissues are treated topically. For solutions in water or aqueous 392

buffer, 50 μL of dosing solutions are applied topically on the RhE tissue and gently spread, 393

if necessary, with a sterile bulb-headed Pasteur pipette (a Pasteur pipette which has been 394

flame-melted to create a small round bulb at one end), or similar device. For solutions in 395

oil, 25 μL of dosing solutions are applied topically on the RhE tissue and gently spread, if 396

necessary, with a sterile bulb-headed Pasteur pipette. If the spreading is not sufficient, 397

consider applying a sterile nylon mesh (circular shape) topically on the tissue as an 398

additional spreading tool (which acts by capillary action to cover the tissue surface). For 399

solvents that may be irritating to skin, the dosing volume should be limited to avoid solvent 400

cytotoxicity. For example, dosing solutions in ethanol, or acetone – olive oil mixture (4:1) 401

should not exceed 20 to 25 μL, since higher volumes may lead to cytotoxicity. 402

403


46. Once dosed, tissues are placed back into the incubator and incubated overnight (18 to 24 404

hours) at standard culture conditions. 405

406

47. On the following day, transfer the tissues into new 6-well plates pre-filled with 0.9 mL of 407

buffered solution (e.g., DPBS or HBSS without phenol red) or 24-well plates pre-filled with 408

0.3 mL of buffered solution. The use of a phenol-red-free buffered salt solution is 409

recommended since irradiation in cell culture medium may lead to increased variability and 410

production of cytotoxic photoproducts (26) 411

412

48. In cases where the test material characteristics may impede or block the irradiation (e.g., 413

dark colored or opaque materials), the dosing dilutions should be removed prior to the 414

irradiation or dark exposure conditions. Sterile cotton swabs soaked in a rinse medium (e.g., 415

DPBS without Ca++ & Mg++ (CMF-DPBS)) may be used to remove the test material prior to 416

the UVA/visible light or dark exposure conditions. Additionally, the dosing dilutions may 417

be washed from the tissues with sterile CMF-DPBS. About 20 washes from a wash bottle 418

are recommended to effectively remove the materials from the tissue surface. If a mesh was 419

used during dosing, ensure that the mesh is removed as part of the rinsing process. The 420

procedures used to remove the dosing dilutions should be documented and presented in the 421

final report. 422

423

49. Irradiation: Irradiate the +Irr plates (covered with lids) for 60 minutes with 1.7 mW/cm2 424

(or equivalent) at room temperature to achieve 6 J/cm2 of simulated sunlight. If the light 425

source generates excess heat and induces condensation under the plate lids, ventilate the 426

plates with a fan. Place the −Irr plates in the dark (e.g., in a box) at room temperature, 427

preferably in the same exposure room as for the tissues being irradiated. Prepare new 6-well 428

plates containing 0.9 mL of warmed (37 ºC) fresh assay medium per well. 429

430

50. After the irradiation is completed, use a wash bottle with sterile CMF-DPBS and rinse each 431

tissue. About 20 washes are needed to effectively remove the materials from the tissue 432

surface. In cases where the tissues were rinsed prior to the irradiation and non-irradiation 433

step, further rinsing is not needed. Transfer all washed inserts to the new plates containing 434

fresh media. The surface of each tissue should be carefully dried using a sterile cotton 435

tipped swab. 436

437

51. Incubate the tissues overnight (18 to 24 hours) at standard culture conditions. 438

439

52. MTT Viability Assay: A 1 mg/mL MTT solution will be prepared, warmed at 37 ºC, and 440

300 μL pipetted into the appropriate wells of a labeled 24-well plate. After the 18 to 24-441

hour incubation, the tissue inserts are removed from the 6-well plates, the bottom of the 442

inserts blotted on sterile gauze or paper towels, and transferred into the appropriate wells of 443

the labeled 24-well MTT plates. The 24-well plates are incubated at standard culture 444

conditions for 3 hours. 445

446

53. After the MTT incubation, the inserts are removed from the 6-well plates, the bottom of the 447

inserts blotted on sterile gauze or paper towels, and transferred into the appropriate wells of 448

new labeled 24-well plates. The tissues are extracted in 2 mL of isopropanol (extraction 449

solution). The 24-well plates will be sealed (e.g., with Parafilm) and the formazan extracted 450

for at least 2 hours at room temperature with gentle shaking on a plate shaker. Alternatively, 451

overnight extraction is also possible. The plates are sealed as described above and extracted 452

at room temperature in the dark, without shaking. Before sampling the extracts, shake for at 453

least 15 minutes on a plate shaker. 454

455

54. After the extraction is completed, the tissue inserts may either be lifted out of the well and 456

the extraction solution decanted into the well from which the insert was taken, or the tissues 457


may be pierced (e.g., with a 20 gauge injection needle) and the extraction allowed to drain 458

into the well from which the insert was taken (the insert can be discarded). The extract will 459

be mixed by pipetting “up and down” at least 3 times until the extraction solution is 460

homogenous. For each tissue, 200 μL aliquots of the extraction solution are pipetted into a 461

labeled 96-well flat bottom microtiter plate. Finally, 200 µL aliquots of isopropanol will be 462

added to the wells designated for the blanks. 463

464

55. The optical density (OD) of the 96-well plate will be determined using a microtiter-plate 465

spectrophotometer using a wavelength between 540 and 595 nm, preferably at 570 nm (with 466

a filter band pass of maximum ± 30 nm). No reference filter reading is required. 467

Alternatively, the absorbance of the formazan extraction samples can be determined using 468

an HPLC/UPLC-spectrophotometry procedure (23). 469

470

471

Cell Viability Calculations 472 473

56. Viability Calculation. The OD values obtained with each test chemical can be used to 474

calculate the percentage of viability relative to the solvent (i.e. negative) control, which is 475

set to 100%. In case HPLC/UPLC-spectrophotometry is used, the percent tissue viability is 476

calculated as percent MTT formazan peak area obtained with living tissues exposed to the 477

test chemical relative to the MTT formazan peak obtained with the concurrent solvent (i.e. 478

negative) control. 479

480

57. The relative viability (or % of Control) of each of the test chemical or positive control-481

treated tissues (+Irr) will be calculated relative to the mean of the appropriate solvent (i.e., 482

negative) control-treated tissues (+Irr). Similarly, the relative viability (or % of Control) of 483

the test article or positive control-treated tissues (−Irr) will be calculated relative to the 484

mean of the appropriate solvent (i.e. negative) control-treated tissues (−Irr). The individual 485

% of Control values are averaged to calculate the mean % of Control (viability) per 486

concentration for each of the +Irr and −Irr exposures. The following equation will be used: 487

488

% 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 =𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑂𝐷 𝑜𝑓 𝑒𝑎𝑐ℎ 𝑇𝑒𝑠𝑡 𝐶ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝑜𝑟 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑇𝑟𝑒𝑎𝑡𝑒𝑑

𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑂𝐷 𝑜𝑓 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒/𝑆𝑜𝑙𝑣𝑒𝑛𝑡 𝐶𝑜𝑛𝑡𝑟𝑜𝑙× 100 489

490

491

492

Corrections for MTT-reducing Materials and Colorants 493

494 58. Optical properties of the test chemical or its chemical action on MTT (e.g., chemicals may 495

prevent or reverse the colour generation as well as cause it) may interfere with the assay 496

leading to a false estimate of viability. This may occur when a specific test chemical is not 497

completely removed from the tissue by rinsing or when it penetrates the epidermis. If a test 498

chemical acts directly on the MTT (e.g., MTT-reducer), is naturally coloured, or becomes 499

coloured during tissue treatment, additional controls should be used to detect and correct for 500

test chemical interference with the viability measurement. Detailed description of how to 501

correct direct MTT reduction and interferences by colouring agents is available in the SOPs 502

for the OECD Validated test methods on skin and eye irritation and corrosion. 503

504

59. To identify direct MTT reducers, each test chemical, at the highest test concentration, 505

should be added to freshly prepared MTT solution. If the MTT mixture containing the test 506

chemical turns blue/purple, the test chemical is presumed to directly reduce MTT and a 507

further functional check on non-viable RhE tissues should be performed, independently of 508

using the standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry 509

procedure. This additional functional check employs killed tissues that possess only 510


residual metabolic activity but absorb the test chemical in a similar way as viable tissues. 511

Each MTT reducing test chemical is applied on at least two killed tissue replicates (e.g., one 512

tissue to be irradiated and one tissue exposed under the dark conditions) at the highest test 513

concentration, which undergo the entire testing procedure to generate a non-specific MTT 514

reduction (NSMTT). 515

516

60. A single NSMTT control is sufficient per test chemical regardless of the number of 517

independent tests/runs performed. The true tissue viability is then calculated as the percent 518

tissue viability obtained with living tissues exposed to the MTT reducer minus the percent 519

non-specific MTT reduction obtained with the killed tissues exposed to the same MTT 520

reducer, calculated relative to the solvent (i.e. negative) control run concurrently to the test 521

being corrected (%NSMTT). 522

523 61. To identify potential interference by coloured test chemicals or test chemicals that become 524

coloured when in contact with water or isopropanol, and to determine the need for 525

additional controls, analysis of the test chemical in water (environment during exposure) 526

and/or isopropanol (extracting solution) should be performed. If the test chemical in water 527

and/or isopropanol absorbs light in the range of 570 ± 30 nm, additional colorant controls 528

should be used. Alternatively, an HPLC/UPLC-spectrophotometry procedure should be 529

used in which case these controls are not required. 530

531

62. When performing the standard absorbance (OD) measurement, each interfering coloured 532

test chemical is applied on at least two viable tissues (e.g., one tissue to be irradiated and 533

one tissue exposed under the dark conditions) at the highest test concentration, which 534

undergoes the entire testing procedure but is incubated with medium instead of MTT 535

solution during the MTT incubation step to generate a non-specific colour (NSCliving) 536

control. The NSCliving control needs to be performed concurrently to the testing of the 537

coloured test chemical and in case of multiple testing, an independent NSCliving control 538

needs to be conducted with each test performed (in each run) due to the inherent biological 539

variability of living tissues. The true tissue viability is then calculated as the percent tissue 540

viability obtained with living tissues exposed to the interfering test chemical and incubated 541

with MTT solution minus the percent non-specific colour obtained with living tissues 542

exposed to the interfering test chemical and incubated with medium without MTT, run 543

concurrently to the test being corrected (%NSCliving). 544

545 63. It is important to note that non-specific MTT reduction and non-specific colour 546

interferences may increase the readouts of the tissue extract above the linearity range of the 547

spectrophotometer. On this basis, each laboratory should determine the linearity range of 548

their spectrophotometer with MTT formazan (CAS # 57360-69-7) from a commercial 549

source before initiating the testing of test chemicals for regulatory purposes. The standard 550

absorbance (OD) measurement using a spectrophotometer is appropriate to assess direct 551

MTT-reducers and colour interfering test chemicals when the ODs of the tissue extracts 552

obtained with the test chemical without any correction for direct MTT reduction and/or 553

colour interference are within the linear range of the spectrophotometer. 554

555 64. For coloured test chemicals which are not compatible with the standard absorbance (OD) 556

measurement due to strong interference with the MTT assay, the alternative HPLC/UPLC-557

spectrophotometry procedure to measure MTT formazan may be employed. The 558

HPLC/UPLC-spectrophotometry system allows for the separation of the MTT formazan 559

from the test chemical before its quantification (36). For this reason, NSCliving or 560

NSCkilled controls are never required when using HPLC/UPLC spectrophotometry, 561

independently of the chemical being tested. NSMTT controls should nevertheless be used if 562

the test chemical is suspected to directly reduce MTT or has a colour that impedes the 563

assessment of the capacity to directly reduce MTT. When using HPLC/UPLC-564


spectrophotometry to measure MTT formazan, the percent tissue viability is calculated as 565

percent MTT formazan peak area obtained with living tissues exposed to the test chemical 566

relative to the MTT formazan peak obtained with the concurrent solvent (i.e. negative) 567

control. For test chemicals able to directly reduce MTT, true tissue viability is calculated as 568

the percent tissue viability obtained with living tissues exposed to the test chemical minus 569

%NSMTT. Finally, it should be noted that direct MTT-reducers that may also be colour 570

interfering, which are retained in the tissues after treatment and reduce MTT so strongly 571

that they lead to ODs (using standard OD measurement) or peak areas (using UPLC/HPLC-572

spectrophotometry) of the tested tissue extracts that fall outside of the linearity range of the 573

spectrophotometer cannot be assessed, although these are expected to occur in only very 574

rare situations. 575

576 65. HPLC/UPLC-spectrophotometry may be used also with all types of test chemicals 577

(coloured, non-coloured, MTT-reducers and non-MTT reducers) for measurement of MTT 578

formazan. Due to the diversity of HPLC/UPLC-spectrophotometry systems, qualification of 579

the HPLC/UPLC-spectrophotometry system should be demonstrated before its use to 580

quantify MTT formazan from tissue extracts by meeting the acceptance criteria for a set of 581

standard qualification parameters based on those described in the U.S. Food and Drug 582

Administration guidance for industry on bio-analytical method validation. 583

584

Criteria for a Valid Test 585 586

66. The following acceptance criteria should be met for a valid test run: 587

The difference in the relative viability values between the two replicate tissues treated 588

with the solvent (i.e. negative) or positive controls should not exceed 20 %. 589

The viability of the solvent (i.e. negative) controls tested in the absence of irradiation 590

should fall within the acceptance range presented in Table 2. 591

The viability of the solvent (i.e. negative) controls tested in the presence of irradiation 592

should result in a viability of ≥80% when compared to the solvent (i.e. negative) 593

controls tested in the absence of irradiation. 594

The positive control should result in a positive prediction. 595

596

67. The following criteria should be met for each of the test substance treatment groups to be 597

evaluable for phototoxic potential: 598

The viability of the test substance-treated tissues in the absence of irradiation should 599

be sufficiently high (for example, >35% viability) to ensure ability to make both 600

phototoxic and not phototoxic predictions at the maximum recommended 601

concentration of 10% (100 mg/mL), or when the maximum concentrations are limited 602

by cytotoxicity, at the highest tolerated dose(s). 603

604

Interpretation of Results and Prediction Model 605 606

68. A chemical is predicted to be phototoxic (or to have phototoxic potential) if the relative 607

viability values for one or more test concentrations treated in the presence of irradiation 608

result in a decrease in viability exceeding 30% when compared to the relative viability 609

values for the same concentrations treated in the absence of irradiation. 610

611

69. A chemical is predicted to be not phototoxic (or to not have phototoxic potential) if none of 612

the relative viability values for the test concentrations treated in the presence of irradiation 613

result in a decrease in viability exceeding 30% when compared to the relative viability 614

values for the same concentrations treated in the absence of irradiation. 615

616

70. If the relative viability for one or more test concentrations treated in the presence of 617

irradiation result in a decrease in viability exceeding 25 % when compared to the relative 618


viability values for the same concentrations treated in the absence of irradiation and 619

provides a difference between two identically treated tissues of > 5%, the test material 620

may have phototoxic potential. If at least one concentration is considered phototoxic (i.e., 621

meets the criteria in paragraph 68), one run will be sufficient to determine phototoxic 622

potential. The experiment should be repeated if none of the concentrations exhibit 623

phototoxic potential. In this case, it is recomended to select a concentration range that is 624

closer to the concentration in which the potentially phototoxic outcome was observed. 625

626

DATA AND REPORTING: 627 628

Data 629 630

71. Quality and quantity of data. Appropriate concentrations which capture the concentration-631

responses in the presence and absence of irradiation should be selected to allow meaningful 632

analysis of the data. Equivocal, borderline, or unclear results should be clarified by further 633

testing. In such cases, modification of experimental conditions (e.g., concentrations tested) 634

should be considered. 635

636

72. For each run, data from individual replicate tissues (e.g., OD values and calculated 637

percentage cell viability data for each test chemical, including classification) should be 638

reported, including data from repeat experiments, as appropriate. In addition, Viability 639

means ± Difference between the duplicate tissues for each run should be reported. Observed 640

interactions with MTT reagent and coloured test chemicals should be reported for each 641

tested chemical. 642

643

Test Report 644

645

73. The test report should include the following information: 646

647

Test Chemical and Control Substances: 648

649

Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS 650

number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as 651

appropriate and practically feasible, etc.; 652

Multi-constituent substance, UVCB and mixture: characterised as far as possible by chemical 653

identity (see above), quantitative occurrence and relevant physicochemical properties of the 654

constituents; 655

Physical appearance, water solubility, and any additional relevant physicochemical properties; 656

Source, lot number if available; 657

Preparation of the test chemical/control substance prior to testing, if applicable (e.g., 658

warming, grinding); 659

Stability of the test chemical, expiration date, or date for re-analysis if known; 660

Storage conditions; 661

Solvent (justification for the choice of solvent; solubility of the test chemical in solvent) 662

663

RhE model and protocol used (and rationale for the choice, if applicable): 664

665

RhE model used (including batch number); 666

Complete supporting information for the specific RhE model used including its performance. 667

This should be provided as a Certificate of Analysis or QC release report by the tissue 668

developer/supplier and may include, but is not limited to; 669

i) Viability; 670

ii) Barrier function; 671


iii) Morphology; 672

iv) Quality controls (QC) of the model 673

674

Reference to historical data of the model. This should include, but is not limited to 675

acceptability of the QC data with reference to historical batch data; 676

Statement of proficiency in performing the test method by testing of the proficiency 677

substances. 678

679

Test Conditions: 680

681

Calibration information for measuring device (e.g., spectrophotometer), wavelength and band 682

pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring 683

device; Description of the method used to quantify MTT formazan; 684

Description of the qualification of the HPLC/UPLC-spectrophotometry system, if applicable; 685

Light source – irradiation conditions: 686

- rationale for selection of the light source used; 687

- manufacturer and type of light source and radiometer; 688

- full spectral irradiance characteristics of the light source; 689

- transmission and absorption characteristics of the filter(s) used; 690

- characteristics of the radiometer and details on its calibration; 691

- distance of the light source from the test system; 692

- UVA irradiance at this distance, expressed in mW/cm2; 693

- duration of the irradiation exposure; 694

- UVA dose (irradiance x time), expressed in J/cm2; 695

- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark. 696

697

Test Procedure: 698

699

Details of the test procedure used (including washing procedures used after exposure period); 700

Doses of test chemical and control substances used; 701

Rationale for selection of concentrations of the test chemical used in the presence and in the 702

absence of irradiation; 703

Type and composition of solvent/vehicle; 704

Duration and temperature of exposure and post-exposure incubation period; 705

Indication of controls used for direct MTT-reducers and/or colouring test chemicals, if 706

applicable; 707

Number of tissue replicates used per test chemical and controls (PC, solvent (i.e. negative) 708

control, and NSMTT, and NSCliving, if applicable); 709

Description of decision criteria/prediction model applied based on the RhE model used; 710

Description of any modifications to the test procedure (including washing procedures). 711

712

Run and Test Acceptance Criteria: 713

714

Acceptance criteria for variability between tissue replicates for positive and solvent (i.e. 715

negative) controls; 716

Acceptance criteria for solvent (i.e. negative) control OD values; 717

Acceptance criteria for the viability of the solvent (i.e. negative) controls in the presence 718

of irradiation relative to those in the absence of irradiation; 719

Acceptance criteria for the positive control. 720

721

Results: 722

723


Tabulation of data for individual test chemical for each run and each replicate 724

measurement including OD or MTT formazan peak area, percent tissue viability, mean 725

percent tissue viability and difference between tissues; 726

If applicable, results of controls used for direct MTT-reducers and/or colouring test 727

chemicals including OD or MTT formazan peak area, %NSMTT, %NSCliving, final 728

corrected relative viability; 729

Results obtained with the test chemical(s) and control substances in relation to the defined 730

run and test acceptance criteria; 731

Description of other effects observed; 732

The derived classification with reference to the prediction model/decision criteria used 733

734

Discussion of the results. 735

736

Conclusions. 737

738

739

740

LITERATURE 741 742

See Last Page. 743


ANNEX 1: Definitions 744

745

Mixture: A mixture or a solution composed of two or more chemicals in which they do not react (4). 746

747

Irradiance: the intensity of ultraviolet (UV) or visible light incident on a surface, measured in W/ m2 748

or mW/ cm2. 749

750

Dose of light: the quantity (= intensity x time) of ultraviolet (UV) or visible radiation incident on a 751

surface, expressed in Joules (= W x s) per surface area, e.g., J/ m2 or J/ cm2. 752

753

UV light wavebands: the designations recommended by the CIE (Commission Internationale de L’754

Eclairage) are: UVA (315-400nm) UVB (280-315nm) and UVC (100-280nm). Other designations 755

are also used; the division between UVB and UVA is often placed at 320nm, and the UVA may be 756

divided into UV-A1 and UV-A2 with a division made at about 340nm. 757

758

Relative tissue viability: tissue viability expressed in relation to solvent (i.e. negative) controls which 759

have been taken through the whole test procedure (either +Irr or -Irr) but not treated with test 760

chemical. 761

762

MEC (Molar Extinction/absorption Coefficient): a constant for any given molecule under a specific 763

set of conditions (e.g., solvent, temperature, and wavelength) and reflects the efficiency with which a 764

molecule can absorb a photon (typically expressed as L⋅mol-1⋅cm-1). 765

766

Phototoxicity: acute toxic response that is elicited after the first exposure of skin to certain chemicals 767

and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic 768

administration of a chemical. 769

770

Accuracy: The closeness of agreement between test method results and accepted reference values. It 771

is a measure of test method performance and one aspect of relevance. The term is often used 772

interchangeably with “concordance” to mean the proportion of correct outcomes of a test method (9). 773

774

Cell viability: Parameter measuring total activity of a cell population e.g., as ability of cellular 775

mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-776

diphenyltetrazolium bromide, Thiazolyl blue), which depending on the endpoint measured and the test 777

design used, correlates with the total number and/or vitality of living cells. 778

779

Chemical: means a substance or a mixture. 780

Concordance: This is a measure of test method performance for test methods that give a categorical 781

result, and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and 782

is defined as the proportion of all chemicals tested that are correctly classified as positive or solvent 783

(i.e. negative). Concordance is highly dependent on the prevalence of positives in the types of test 784

chemical being examined (9). 785

786

HPLC: High Performance Liquid Chromatography. 787

788

IATA: Integrated Approach on Testing and Assessment 789

790

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium 791

bromide. 792

793

Negative Control: see Solvent Control 794

795

NSCkilled: Non-Specific Colour in killed tissues. 796


797

NSCliving: Non-Specific Colour in living tissues. 798

799

NSMTT: Non-Specific MTT reduction. 800

801

Performance standards (PS): Standards, based on a validated test method, that provide a basis for 802

evaluating the comparability of a proposed test method that is mechanistically and functionally 803

similar. Included are; (i) essential test method components; (ii) a minimum list of Reference 804

Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the 805

validated test method; and (iii) the comparable levels of accuracy and reliability, based on what was 806

obtained for the validated test method, that the proposed test method should demonstrate when 807

evaluated using the minimum list of Reference Chemicals (9). 808

809

Positive Control (PC): a replicate containing all components of a test system and treated with a 810

substance known to induce a positive response. To ensure that variability in the positive control 811

response across time can be assessed, the magnitude of the positive response should not be excessive. 812

813

Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful 814

and useful for a particular purpose. It is the extent to which the test correctly measures or predicts the 815

biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a 816

test method (9). 817

818

Reliability: Measures of the extent that a test method can be performed reproducibly within and 819

between laboratories over time, when performed using the same protocol. It is assessed by calculating 820

intra- and inter-laboratory reproducibility (9). 821

822

Replacement test: A test which is designed to substitute for a test that is in routine use and accepted 823

for hazard identification and/or risk assessment, and which has been determined to provide equivalent 824

or improved protection of human or animal health or the environment, as applicable, compared to the 825

accepted test, for all possible testing situations and chemicals (9). 826

827

Run: A run consists of one or more test chemicals tested concurrently with a solvent (i.e. negative) 828

control and with a PC. 829

830

Sensitivity: The proportion of all positive/active test chemicals that are correctly classified by the test. 831

It is a measure of accuracy for a test method that produces categorical results, and is an important 832

consideration in assessing the relevance of a test method (9). 833

834

Solvent Control: A replicate containing all components of a test system except for the test chemical, 835

but including the solvent that is used. It is used to establish the baseline response for the samples 836

treated with the test chemical dissolved in the same solvent, and in this Test Method is used as a 837

negative control in the data analyses. This sample is processed with test chemical-treated samples and 838

other control samples. 839

840

Specificity: The proportion of all negative/inactive test chemicals that are correctly classified by the 841

test. It is a measure of accuracy for a test method that produces categorical results and is an important 842

consideration in assessing the relevance of a test method (9). 843

844

Test chemical: is the term “test chemical” is used to refer to what is being tested. 845

846

UPLC: Ultra-High Performance Liquid Chromatography. 847

848

UVCB: substances of unknown or variable composition, complex reaction products or biological 849

materials 850


851

ANNEX 2: Use of the RhE Phototoxicity Test for evaluating 852

formulations at “end-use” concentrations 853

854

Whereas this Test Guideline was designed to evaluate the phototoxicity potential of individual 855

chemicals by optimizing the test conditions and solvents selected for chemical hazard assessment, the 856

test method has also been utilized to evaluate the phototoxic/photoirritant effects of complex mixtures 857

and formulations at “end-use” concentrations (28)(29). Product formulations or complex mixtures 858

intended for skin application, or expected to come in contact with skin, may be tested undiluted at a 859

single “end user” concentration. The same mechanistic events are relevant for measuring phototoxic 860

and cytotoxic effects in test substance-treated RhE tissues in the presence and absence of exposure to 861

simulated sunlight, except that the testing of formulations at the end-user concentration presents all 862

ingredients of a complex formulation topically onto the RhE tissue in a manner that more closely 863

models the topical skin exposures in vivo. The testing of undiluted formulations and complex mixtures 864

is justified by the following: 865

it allows for the topical application of individual ingredient chemicals at the maximum doses 866

likely encountered in vivo; 867

it models the permeation kinetics of the individual ingredient chemicals given the 868

thermodynamic effects of the vehicle and all of the ingredients; 869

it allows modeling of synergistic and antagonistic effects of all of the formulation ingredients, 870

in the presence and absence of irradiation. 871

Thus under the test conditions, ingredient chemicals within a formulation that permeate into the RhE 872

tissue during the 18 to 24-hour exposure period are bioavailable at the time of irradiation. 873

Subsequently, the phototoxicity potential of the formulation is evaluated as a whole by the relative 874

reduction in viability of treated cultures in the presence as compared to the absence of simulated 875

sunlight. Therefore, the purpose of the approach then is to evaluate the phototoxicity potential of the 876

formulation under end-use conditions, rather than to determine the phototoxicity potential of a 877

specific chemical. 878

The testing of undiluted complex mixtures and end-use formulations follows the same general 879

methodology described in the Test Guideline, with the following exceptions: 880

since formulations are tested undiluted, no dilution series are prepared or tested; 881

no evaluation of test substance solubility is conducted; 882

no test substance solvents are utilized; 883

therefore, the negative (i.e., solvent) control will reflect the solvent used for the preparation 884

of the positive control. 885

886

In addition, the following methods are implemented for testing undiluted end-use formulations: 887

888

before any testing on the viable reconstructed human tissues is performed, it is recommended 889

to perform the evaluation of the test substance for interference with the measured endpoint 890


(MTT assay). The undiluted formulations are evaluated in the same manner that an individual 891

chemical is evaluated. 892

the standard positive control (and solvent control) for the test method described in paragraph 893

35 will be utilized to validate the test method run. No recommendation is made for users to 894

consider “spiking” a known phototoxic reference chemical into the test formulation to 895

validate the run, since the formulation may readily interact with, or impact the permeation 896

kinetics of the reference chemical, resulting in notably different cytotoxicity and 897

phototoxicity results relative to those obtained for the reference chemical in an optimized 898

solvent. In such cases, a non-phototoxic result should not automatically invalidate the test run. 899

900

Dose Application: 901

902

the RhE tissues are treated topically. Since end-use formulations for topical application are 903

often viscous, 50 μL of the undiluted formulations are applied topically on the RhE tissue 904

using a positive displacement pipette, and the dose is gently spread, if necessary, using the 905

positive displacement pipette tip. Alternatively, the dose may be spread with a sterile bulb-906

headed Pasteur pipette, or similar device. 907

since formulations may typically be opaque or darkly colored, these materials should 908

routinely be adequately rinsed off of the tissues prior to irradiation to avoid possible 909

interference with the photo-irradiation. Sterile cotton swabs soaked in a rinse medium (e.g., 910

DPBS without Ca++ & Mg++ (CMF-DPBS)) may be used to remove the test material prior to 911

the UVA/visible light or dark exposure conditions. Additionally, the dosing dilutions may be 912

washed from the tissues with sterile CMF-DPBS. About 20 washes from a wash bottle are 913

recommended to effectively remove the materials from the tissue surface. 914

915

Criteria for a Valid Test: 916 917

The following acceptance criteria should be met for a valid test run: 918

the difference in the relative viability values between the two replicate tissues treated 919

with the solvent (i.e. negative) or positive controls should not exceed 20 %. 920

the viability of the solvent (i.e. negative) controls tested in the absence of irradiation 921

should fall within the acceptance range presented in Table 2. 922

the viability of the solvent (i.e. negative) controls tested in the presence of irradiation 923

should result in a viability of ≥80% when compared to the solvent (i.e. negative) 924

controls tested in the absence of irradiation. 925

the positive control should result in a positive prediction. 926

927

The following criteria should be met for the test formulation to be evaluable for phototoxic 928

potential: 929

the viability of the test formulation-treated tissues in the absence of irradiation should 930

be sufficiently high (for example, >35% viability) to ensure ability to make both 931

phototoxic and not phototoxic predictions. 932

933


ANNEX 3 934

935

Figure 1. Spectral power distribution of a filtered solar simulator. 936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

Figure 1 gives an example of an acceptable spectral power distribution of a filtered solar simulator. It 955

is from the doped metal halide source used in the validation trial of the 3T3 NRU PT as well as pre-956

validation of the EpiDerm Phototoxicity test and in most of the follow-up studies. The effect of two 957

different filters and the additional filtering effect of the lid of a 96-well cell culture plate are shown. 958

The H2 filter was only used with test systems that can tolerate a higher amount of UVB (skin model 959

test and red blood cell photohemolysis test). In the 3T3 NRU-PT the H1 filter was used. The figure 960

shows that additional filtering effect of the plate lid is mainly observed in the UVB range, still leaving 961

enough UVB in the irradiation spectrum to excite chemicals typically absorbing in the UVB range, 962

like Amiodarone. 963

964

Figure 2. Irradiation sensitivity of RhE (as measured in the UVA range) 965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

This figure presented in Liebsch et al (1999) shows the responses of tissues exposed to increasing 981

concentrations of UVA irradiation relative to non-irradiated tissues. Relative viability was determined 982

using the MTT conversion assay. Each box represents the mean of 12 tissues evaluated over four 983

independent experiments. The tissues tolerated a dose of 6 J/cm2 without excess cytotoxic effects. 984

985


ANNEX 4: Considerations in the selection of test chemical solvents 986

987

Solvents / vehicles: 988

During the development and pre-validation study (Liebsch, et al., 1997 and 1999a), sesame seed oil 989

was chosen as a solvent and vehicle for chemicals which could not be sufficiently dissolved in water. 990

Several other solvents were investigated by the other laboratories participating in the pre-validation, 991

but the sesame seed oil was chosen for the final experiments. In addition to oily solvents, ethanol and 992

a mixture of acetone:olive oil were suggested for materials that could not be readily solubilised in 993

water or oil (Jones, et al., 1999 and Liskova, et al., 2018). 994

It is of importance to select a solvent that will sufficiently transmit the full spectrum of the simulated 995

sunlight (i.e., the solvent should not show appreciable absorption within the simulated sunlight 996

spectrum). Furthermore, the recommended dosing volume of 50 µL should not be exceeded, since 997

excessive volumes of solvent/vehicle on the tissue surface may create a photo-protective layer. 998

The photopotency (i.e. the phototoxic strength) of chemicals may be modulated by the solvent/vehicle 999

as demonstrated in experiments obtained for Chlopromazine in oily and aqueous solutions (Liebsch, 1000

et al., 1997) or with Anthracene tested in oily and ethanolic solutions (Liebsch, et al., 1997 and 1001

Liskova, et al., 2018). 1002

1003

Absorption / transmission spectra of three oils and DMI 1004

0

20

40

60

80

100

120

250 300 350 400 450 500Wavelength [nm]

Tra

nsm

issio

n

Sesame Oil (P&G)

Olive Oil (P&G)

Arlasolve DMI

Mineral Oil

1005 1006

Source: Manfred Liebsch, Prevalidation of the "EpiDerm™ 1007

Phototoxicity Test" FINAL REPORT (Phases I, II, III) 1008 1009

1. Liebsch, M., Barrabas, C., Traue, D. and Spielmann, H. (1997) Entwicklung eines neuen in vitro Tests auf dermale Phototoxizität 1010 mit einem Modell menschlicher Epidermis (EpiDermTM). ALTEX 14: 165 - 174 1011 1012

2. Jones, P., King.A., Lovel., W., Earl, L (1999). Phototoxicity tesing using 3D Reconstructed Human skin models. In Alternatives 1013 to Animal Testing II: Proceedings of the second international scientific conference organised by the European Cosmetic Industry, 1014 Brussels, Belgium (ed. D. Clark, S. Lisansky & R. Macmillan), pp. Newbury, UK: CPL Press 1015

1016

3. Líšková, A., Letašiová, S., Jantová, S., Brezová, V. and Kandárová, H. (2020) “Evaluation of phototoxic and cytotoxic potential 1017 of TiO2 nanosheets in a 3D reconstructed human skin model”, ALTEX - Alternatives to animal experimentation, 37(3), pp. 441-1018 450. doi: 10.14573/altex.1910012 1019 1020

4. Liebsch, M. (1999b). Prevalidation of the "EpiDerm™ Phototoxicity Test" FINAL REPORT (Phases I, II, III). 31 pages 1021 1022


1023

REFERENCES 1024


1 Kandárová, H., Raabe, H., Hilberer, A., Choksi, N., and Allen D. Retrospective review on in vitro

phototoxicity data generated in 3D skin models to support development of new OECD test Guideline.

Society of Toxicology Annual Meeting, Virtual Event, 12-26 March 2021.

2 Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Gerberick, G.F., Cruse, L., Diembeck, W.,

Pfannenbecker, U., Spieker, J., Holzhütter, H.G., Brantom, P., Aspin, P., and Southee, J. (1999). In

Alternatives to Animal Testing II: Proceedings of the second international scientific conference

organised by the European Cosmetic Industry, Brussels, Belgium (ed. D. Clark, S. Lisansky & R.

Macmillan), pp. 160–166. Newbury, UK: CPL Press

3 Lovell, W.W. (1993). A scheme for in vitro screening of substances for photoallergenic potential.

Toxic. In Vitro 7: 95-102.

4 Santamaria, L., and Prino, G. (1972). List of the photodynamic substances. In “Research Progress in

Organic, Biological and Medicinal Chemistry” Vol. 3 part 1. North Holland Publishing Co.

Amsterdam. p XI-XXXV.

5 Spielmann, H., Lovell, W.W., Hölzle, E., Johnson, B.E., Maurer, T., Miranda, M.A., Pape, W.J.W.,

Sapora, O., and Sladowski, D. (1994). In vitro phototoxicity testing: The report and recommendations

of ECVAM Workshop. ATLA, 22, 314-348.

6 Spikes, J.D. (1989). Photosensitization. In “The science of Photobiology” Edited by K.C. Smith.

Plenum Press, New York. 2nd edition, p 79-110.

7 Bauer, D., Averett, L.A., De Smedt, A., Kleinman, M.H., Muster, W., Pettersen, B.A., and Robles,

C. (2014). Standardized UV-vis spectra as the foundation for a threshold-based, integrated

photosafety evaluation. Regul Toxicol Pharmacol, 68: 70-75.

8 ICH S10 Photosafety Evaluation of Pharmaceuticals. Guidance for Industry. January 2015.

https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm06500 7.htm

9 OECD (1997) Environmental Health and Safety Publications, Series on Testing and Assessment No.

7 “Guidance Document On Direct Phototransformation Of Chemicals In Water” Environment

Directorate, OECD, Paris

10 Oesch, F., Fabian, E., Guth, K., and Landsiedel, R. Xenobiotic-metabolizing enzymes in the skin of

rat, mouse, pig, guinea pig, man, and in human skin models. Arch Toxicol. 2014; 88(12):2135–2190.

doi:10.1007/s00204-014-1382-8

11 Ceridono, M., Tellner, Par, Bauer, D., Barroso, J., Alépée, N., Corvi, R., De Smedt, A., Fellows,

M.D., Gibbs, N.K., Heisler, E., Jacobs, A., Jirova, D., Jones, D., Kandárová, H., Kasper, P., Akunda,

J.K., Krul, C., Learn, D., Liebsch, M., Lynch, A.M., Muster, W., Nakamura, K., Nash, J.F.,

Pfannenbecker, U., Phillips, G., Robles, C., Rogiers, V., Van De Water, F., Liminga, U.W., Vohr,

H.W., Wattrelos, O., Woods, J., Zuang, V., Kreysa, J., and Wilcox, P. (2012) The 3T3 neutral red

uptake phototoxicity test: practical experience and implications for phototoxicity Testing – The report

of an ECVAM-EFPIA workshop. Reg Tox Pharm. 63: 480- 488.

12 Jones, P., King, A., Lovell, W., and Earl, L. Phototoxicity testing using 3-D reconstructed human

skin models. In: Clark D, Lisansky S, Macmillan R, editors. Alternatives to animal testing II:

proceedings of the second international scientific conference organised by the European cosmetic

Industry, Brussels, Belgium. Newbury, UK: CPL Press; 1999. p. 138–41.

13 Spielmann et al


14 INVITTOX Protocol 121. EpiDerm™ Phototoxicity Assay. ECVAM DB-ALM; 1999.

http://ecvam-dbalm.jrc.ec.europa.eu/

15Kandárová,H., and Liebsch, M. (2017) The EpiDerm™ Phototoxicity Test (EpiDerm™ H3D-PT).

Book Chapter In: Alternatives for Dermal Toxicity Testing, Editors: Chantra Eskes, Erwin van Vliet,

Howard I. Maibach. Springer. 483-503.

16 Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to

Proliferation and Cytotoxicity Assays, J. Immunol. Methods 65, 55- 63.

17 Alépée, N., Barroso, J., De Smedt, A., De Wever, B., Hibatallah, J., Klaric, M., Mewes, K.R.,

Millet, M., Pfannenbecker, U., Tailhardat, M., Templier, M., and McNamee, P. (2015) Use of

HPLC/UPLC-Spectrophotometry for Detection of MTT Formazan in In vitro Reconstructed Human

Tissue (RhT)-Based Test Methods Employing the MTT Assay to Expand their Applicability to

Strongly Coloured Test Chemicals. Toxic. In vitro. 29(4), 741-761.

18Sheehan, D., Pidathala, A., and Hilberer, A. Evaluation of New Solvents for the Use in the Multi‐

Dose Reconstructed Human EpiDermis (RhE) Phototoxicity Assay. Society of Toxicology Annual

Meeting New Orleans, LA USA, 13-17 March 2016

19 Lambert, L.A, Wamer, W.G., and Kornhauser, A. (1996) Animal models for phototoxicity testing.

In “Dermatotoxicology”, edited by F.N. Marzulli and H.I. Maibach. Taylor & Francis, Washington

DC. 5th Edition, p 515-530.

20 Lambert, L.A, Wamer, W.G., and Kornhauser, A. (1996) Animal models for phototoxicity testing.

In “Dermatotoxicology”, edited by F.N. Marzulli and H.I. Maibach. Taylor & Francis, Washington

DC. 5th Edition, p 515-530.

21 ISO 10977. (1993). Photography - Processed photographic colour films and paper prints - Methods

for measuring image stability.

22 Líšková, A., Letašiová, S., Jantová, S., Brezová, V., and Kandárová, H. (2020) “Evaluation of

phototoxic and cytotoxic potential of TiO2 nanosheets in a 3D reconstructed human skin

model”, ALTEX - Alternatives to animal experimentation, 37(3), pp. 441-450. doi:

10.14573/altex.1910012.

23 All general references to the HPLC methods

24 OECD. (2019). OECD Guideline for the Testing of Chemicals (No. 439): In Vitro Skin Irritation:

Reconstructed Human Epidermis Test Method. Organisation for Economic Cooperation and

Development, Paris.

25 OECD. (2019). OECD Guideline for the Testing of Chemicals (No. 431): In Vitro Skin Corrosion:

Reconstructed Human Epidermis Test Method. Organisation for Economic Cooperation and

Development, Paris.

26 Stoien, JD, and Wang, RJ. Effect of near-ultraviolet and visible light on mammalian cells in culture

II. Formation of toxic photoproducts in tissue culture medium by blacklight. Proc Natl Acad Sci U S

A. 1974;71(10):3961-3965. doi:10.1073/pnas.71.10.3961

27 Kejlová, K., Jírová, D., Bendová, H., Kandárová, H., Weidenhoffer, Z., Kolářová, H., and Liebsch,

M. (2007). Phototoxicity of Bergamot oil assessed by in vitro techniques in combination with human

patch tests. Toxic. in Vitro 21, p. 1298–1303

http://ecvam-dbalm.jrc.ec.europa.eu/


28 Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D., and Spielmann, H.

(2005). Skin irritation in vitro: EpiDerm test protocol developed and optimised for an ECVAM

validation study on skin irritation testing of chemicals. 5th World Congress on Alternatives & Animal

Use in the Life sciences. Session 5.3: Progress and Needs for Developing and Validating Alternatives

for Dermal Toxicity Testing. Berlin, August 21–25, 2005. Abstract in : ALTEX 22, Special Issue

2005, page 156, ISSN: 0946-7785

29 Ritacco, G., and Api, A.M. Stepwise Testing of Potentially Phototoxic Fragrance Materials. Society

of Toxicology Annual Meeting Baltimore, MD USA, 12-17 March 2017

30 Ritacco, G., and Api, A.M. Phototoxicity of ethyl vanillin: comparison of two in vitro methods.

Society of Toxicology Annual Meeting New Orleans, LA USA, 13-17 March 2016

Documents

-Draft OECD GUIDELINE FOR TESTING OF CHEMICALS